The major goal of the proposed research is a detailed underestanding of the forces primarily responsible for determining the long-range structure of DNA. Such an understanding will aid in analyzing the means by which DNA is organized into compact structures (e.g., chromosomes and viral particles) as well as the mechanisms by which DNA structure is altered by small ligands (e.g., intercalating drugs), DNA binding proteins, and disease processes which later its chemistry) (e.g., base removal, single-strand nicking, etc.). A knowledge of such mechanisms is essential for the broader understanding of the regulation of gene expression. The primary aim of this application is to identify those features of DNA which contribute to its long-range order. This examination will be carried out in a systematic fashion by varying the DNA sequence, the integrity of its phosphodiester backbone, etc. My experimental approach comprises two parts; namely, (1) the use of recombinant DNA cloning methodologies to produce a wide variety of DNA molecules of precisely defined sequence and length, and (2) the use of two independent, self-consistent experimental methods for measuring the flexibility of DNA. The first method consists of the measurement of rotational relaxation times of DNA molecules of interest by following the field-free decay of birefringence (TEB). The second method consists of the measurement of the rates of formation of small circles (catalyzed by T4 DNA ligase). The second aim of this application is to further characterize the electrostatic contribution to the rigidity of DNA at low ionic strengths. The third aim of this application is to characterize the pathway of interconversion between B-form and Z-form DNA by performing kinetic TEB measurements, utilizing a prototype stopped-flow TEB instrument.